CLE RNAseq analysis QC
Miles Smith
08 October, 2020
Data processing of RNAseq data
Overview of sample characteristics:
## 191 samples selected for analysis## 191 files found## All files match a metadata entry.## All expected files found.Sample QC filtering
Samples with a PC1 > 2 (the red lines) and/or PC2 zscore > 2.5 (the green lines) were dropped from analysis. Points in red represent samples that were dropped.
## Warning: `arrange_()` is deprecated as of dplyr 0.7.0.
## Please use `arrange()` instead.
## See vignette('programming') for more help
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_warnings()` to see where this warning was generated.| sample_name | pt_ref | subject_ref | study | subject_id | process_ref | visit_ref | sample_type | visit_date | sample_alias | age | ethnicity | sex | disease_class | reason_group | match_group | random_seed | plate_id | rin | concentration | cell_type | ifc |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| AC027_22 | BPID|023300 | OCRD|1453 | OCRD | 1453 | OCRD|1453|2017-02-24 | OCRD|1453|2017-02-23 | B Cell RNA | 2017-02-23 | S01453-V08702 | 50 | AI | Female | RA | RA-AI | BPID|020101 | 0.0047414 | RNA NARCH_1 | 9.3 | 2.8 | b_cell | IFC1-1970031135 |
| AC060_22 | BPID|013709 | CE|541425 | CE | 541425 | CE|541425|2010-03-29 | CE|541425|2010-03-29 | B Cell RNA | 2010-03-29 | none | 29 | AA | Female | control | CTRL-AA | BPID|023080 | 0.9362812 | RNA NARCH_1 | 8.9 | 2.8 | b_cell | IFC1-1970031135 |
| AC063_32 | BPID|014195 | CE|541936 | CE | 541936 | CE|541936|2010-06-23 | CE|541936|2010-06-22 | T Cell RNA | 2010-06-22 | none | 48 | AA | Female | control | CTRL-AA | BPID|020101 | 0.6564617 | RNA NARCH_2 | 7.9 | 2.8 | t_cell | IFC3-1970031192 |
Overall correlation between samples
Data quality
MA-plot
This MA plot is of shrunken log2 fold changes (using the apeglm package), in which actual fold changes are adjusted to compensate for the high variablity of genes with few counts.
Dots in red indicate a significant expression difference between Control and SLE.
Dispersion estimates
K-means clustering optimization
To determine the optimal k, the gap statistic was calculated for k = (3,20) (k = 2 was ignored because it just returns the two disease clusters). A higher coefficient means better intracluster simliarity and intercluster differences.
Sample Sanity check
To determine if there is a gross mixup of samples, examine the expression of sex-specific transcripts to ensure that only female-subject samples express “XIST” and male-subject samples "DDX3Y:
These appear to be correct.
## ─ Session info ───────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────
## setting value
## version R version 4.0.0 (2020-04-24)
## os Ubuntu 18.04.4 LTS
## system x86_64, linux-gnu
## ui X11
## language (EN)
## collate en_US.UTF-8
## ctype en_US.UTF-8
## tz Etc/UTC
## date 2020-10-08
##
## ─ Packages ───────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────
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## tidyposterior 0.0.2 2018-11-15 [1] RSPM (R 4.0.0)
## tidypredict 0.4.5 2020-02-10 [1] RSPM (R 4.0.0)
## tidyr * 1.1.0 2020-05-20 [1] RSPM (R 4.0.0)
## tidyselect * 1.1.0 2020-05-11 [1] RSPM (R 4.0.0)
## tidytext 0.2.4 2020-04-17 [1] RSPM (R 4.0.0)
## tidyverse * 1.3.0 2019-11-21 [1] RSPM (R 4.0.0)
## timeDate 3043.102 2018-02-21 [1] RSPM (R 4.0.0)
## tokenizers 0.2.1 2018-03-29 [1] RSPM (R 4.0.0)
## triebeard 0.3.0 2016-08-04 [1] RSPM (R 4.0.0)
## tune * 0.1.0 2020-04-02 [1] RSPM (R 4.0.0)
## tweenr 1.0.1 2018-12-14 [1] RSPM (R 4.0.0)
## tximport * 1.16.1 2020-06-05 [1] Bioconductor
## txtq 0.2.0 2019-10-15 [1] RSPM (R 4.0.0)
## urltools 1.7.3 2019-04-14 [1] RSPM (R 4.0.0)
## uuid 0.1-4 2020-02-26 [1] RSPM (R 4.0.0)
## uwot * 0.1.8 2020-03-16 [1] RSPM (R 4.0.0)
## vctrs 0.3.1 2020-06-05 [1] RSPM (R 4.0.0)
## viridis * 0.5.1 2018-03-29 [1] RSPM (R 4.0.0)
## viridisLite * 0.3.0 2018-02-01 [1] RSPM (R 4.0.0)
## webshot 0.5.2 2019-11-22 [1] RSPM (R 4.0.0)
## WGCNA * 1.69 2020-02-28 [1] RSPM (R 4.0.0)
## withr 2.2.0 2020-04-20 [1] RSPM (R 4.0.0)
## workflows * 0.1.1 2020-03-17 [1] RSPM (R 4.0.0)
## xfun 0.14 2020-05-20 [1] RSPM (R 4.0.0)
## XML 3.99-0.3 2020-01-20 [1] RSPM (R 4.0.0)
## xml2 1.3.2 2020-04-23 [1] RSPM (R 4.0.0)
## xtable 1.8-4 2019-04-21 [1] RSPM (R 4.0.0)
## xts 0.12-0 2020-01-19 [1] RSPM (R 4.0.0)
## XVector 0.28.0 2020-04-27 [1] Bioconductor
## yaml 2.2.1 2020-02-01 [1] RSPM (R 4.0.0)
## yardstick * 0.0.6 2020-03-17 [1] RSPM (R 4.0.0)
## zip 2.0.4 2019-09-01 [1] RSPM (R 4.0.0)
## zlibbioc 1.34.0 2020-04-27 [1] Bioconductor
## zoo 1.8-8 2020-05-02 [1] RSPM (R 4.0.0)
##
## [1] /usr/local/lib/R/site-library
## [2] /usr/local/lib/R/library